Phenotypic plasticity of Escherichia coli upon exposure to physical stress induced by ZnO nanorods.

Evolution of bacteria to selective chemical pressure (e.g. antibiotics) is well studied in contrast to the influence of physical stressors. Here we show that instantaneous physical stress in a homogeneous environment (without concentration gradient) induces fast adaptation of Escherichia coli. We exposed E. coli to a large number of collisions of around 105 per bacterium per second with sharp ZnO nanorods. The pressure exerted on the bacterial cell wall was up to 10 GPa and induced phenotype changes. The bacteria's shape became more spherical, the density of their periplasm increased by around 15% and the average thickness of the cell wall by 30%. Such E. coli cells appeared almost as Gram-positive bacteria in the standard Gram staining. Additionally, we observed a combination of changes occurring at the genomic level (mutations identified in form of single nucleotide polymorphisms) and down-regulation of expression of 61 genes encoding proteins involved in ß-oxidation of fatty acids, glycolysis, the citric acid cycle, as well as uptake of amino acids and enzyme cofactors. Thus, we show that bacteria undergo phenotypic changes upon instantaneous, acute physical stress without any obviously available time for gradual adaptation.

Generation of Transgenic Porcine Fibroblast Cell Lines Using Nanomagnetic Gene Delivery Vectors.

The transgenic process allows for obtaining genetically modified animals for divers biomedical applications. A number of transgenic animals for xenotransplantation have been generated with the somatic cell nuclear transfer (SCNT) method. Thereby, efficient nucleic acid delivery to donor cells such as fibroblasts is of particular importance. The objective of this study was to establish stable transgene expressing porcine fetal fibroblast cell lines using magnetic nanoparticle-based gene delivery vectors under a gradient magnetic field. Magnetic transfection complexes prepared by self-assembly of suitable magnetic nanoparticles, plasmid DNA, and an enhancer under an inhomogeneous magnetic field enabled the rapid and efficient delivery of a gene construct (pCD59-GFPBsd) into porcine fetal fibroblasts. The applied vector dose was magnetically sedimented on the cell surface within 30 min as visualized by fluorescence microscopy. The PCR and RT-PCR analysis confirmed not only the presence but also the expression of transgene in all magnetofected transgenic fibroblast cell lines which survived antibiotic selection. The cells were characterized by high survival rates and proliferative activities as well as correct chromosome number. The developed nanomagnetic gene delivery formulation proved to be an effective tool for the production of genetically engineered fibroblasts and may be used in future in SCNT techniques for breeding new transgenic animals for the purpose of xenotransplantation.

Cytotoxicity of thermo-responsive polymeric nanoparticles based on N-isopropylacrylamide for potential application as a bioscaffold.

Polymeric nanoparticles based on poly-N-isopropylacrylamide (pNiPAM NPs) and their bio-medical applications have been widely investigated in recent years. These tunable nanoparticles are considered to be great candidates for drug delivery systems, biosensors and bioanalytical devices. Thus, the biocompatibility and toxicity of these nanoparticles is clearly a crucial issue. In this work, the cytotoxicity of thermo-responsive pNiPAM nanoparticles was studied, followed by a detailed analysis of the NPs morphology in growing cell cultures and their 3D structure. Cytotoxic examination was conducted for two cell cultures - HeLa (cervical cancer cell line) and HeK293 (human embryonic kidney cell line), employing MTT (3-4, 5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide) assay and viability tests. We used Cryo-SEM (scanning electron microscopy) and fluorescence microscopy (IN Cell Analyzer) in order to investigate the morphological structure of the polymer network. We show that pNiPAM nanoparticles do not exhibit any cytotoxicity effects on the investigated cell lines. Additionally, we report that the pNiPAM nanoparticle based scaffold promotes cell growth.

Intrinsically disordered and pliable Starmaker-like protein from medaka (Oryzias latipes) controls the formation of calcium carbonate crystals.

Fish otoliths, biominerals composed of calcium carbonate with a small amount of organic matrix, are involved in the functioning of the inner ear. Starmaker (Stm) from zebrafish (Danio rerio) was the first protein found to be capable of controlling the formation of otoliths. Recently, a gene was identified encoding the Starmaker-like (Stm-l) protein from medaka (Oryzias latipes), a putative homologue of Stm and human dentine sialophosphoprotein. Although there is no sequence similarity between Stm-l and Stm, Stm-l was suggested to be involved in the biomineralization of otoliths, as had been observed for Stm even before. The molecular properties and functioning of Stm-l as a putative regulatory protein in otolith formation have not been characterized yet. A comprehensive biochemical and biophysical analysis of recombinant Stm-l, along with in silico examinations, indicated that Stm-l exhibits properties of a coil-like intrinsically disordered protein. Stm-l possesses an elongated and pliable structure that is able to adopt a more ordered and rigid conformation under the influence of different factors. An in vitro assay of the biomineralization activity of Stm-l indicated that Stm-l affected the size, shape and number of calcium carbonate crystals. The functional significance of intrinsically disordered properties of Stm-l and the possible role of this protein in controlling the formation of calcium carbonate crystals is discussed.